Mono-ADP-ribosylation is a post-translational modification of proteins in which the ADP-ribose moiety of NAD is transferred to protein and is responsible for the toxicity of some bacterial toxins (e.g., cholera toxin, pertussis toxin). Like cholera toxin, some mammalian ADP?ribosyltransferases specifically use the guanidino group of arginine as an ADP-ribose acceptor. Five mammalian NAD: arginine ADP-ribosyltransferases (ART) were cloned from various tissues. ART1 is a cell surface protein, linked through a glycosylphosphatidylinositol (GPI) anchor. The transferases appear to be selectively expressed in mammalian tissues. ART-1 is found in skeletal and cardiac muscle and lymphoid cells, ART-2 in lymphocytes, ART-4 in spleen and ART-5 in testis.The ADP-ribosyltransferases are characterized by a core catalytic domain that is conserved among the mammalian and bacterial enzymes. To define the requirements for ADP-ribosyltransferase activity, truncation mutants were made from the murine homologue of ART-1. This transferase catalyzes the ADP-ribosylation of arginine, and similar small guanidino compounds, as well as peptides and proteins containing arginine residues. In the absence of a guanidino acceptor for ADP-ribose, the transferase hydrolyzes NAD to ADP-ribose and nicotinamide, but at a much slower rate than arginine ADP-ribosylation. Removal of the signal sequences at the amino terminus, which is required for export into the endoplasmic reticulum, and at the carboxy terminus, which is necessary for addition of the GPI anchor, resulted in an active NAD:arginine ADP-ribosyltransferase. Further deletion of sequence from the amino terminus resulted in a significant loss of transferase activity, measured by ADP-ribosylation of both proteins and free arginine. In contrast, deletion of the amino terminal region of the protein increased NAD glycohydrolase activity, suggesting that the amino terminus has an inhibitory effect on abortive hydrolysis of NAD. In contrast, removal of amino acids at the carboxy terminus, which are closer to the putative catalytic site, resulted in a loss of both transferase and glycohydrolase activities.